Mountain Pine Beetle Mutualist Leptographium Longiclavatum Presence in the Southern Rocky Mountains During a Record Warm Period

DOI 10.12905/0380.sydowia70-2018-0001 Published online 30 April 2018

Mountain pine beetle mutualist Leptographium longiclavatum presence in the southern Rocky Mountains during a record warm period

Javier E. Mercado1,* & Beatriz Ortiz-Santana2

1 Rocky Mountain Research Station, 240 W Prospect Rd., Fort Collins, CO 80526 2 Northern Research Station, One Gifford Pinchot Dr., Madison, WI 53726 * e-mail: [email protected]

Mercado J.E. & Ortiz-Santana B. (2018) Mountain pine beetle mutualist Leptographium longiclavatum presence in the southern Rocky Mountains during a record warm period. – Sydowia 70: 1–10. We studied blue-stain fungi (Ophiostomataceae: Ophiostomatales) of mountain pine beetle in declining epidemic populations affecting three pine species in Colorado. Using morphological and molecular characterizations, we determined the presence of the mutualist L. longiclavatum in the southern Rocky Mountains of Colorado, where it was as common as the warm temperature adapted O. montium within the insect’s specialized maxillary mycangium. The species was more prevalent than its “sibling species” G. clavigera which is the the common mycangial mutualist documented in USA populations. Findings were made during a two-year period including the warmest year on record in the state (i. e., 2012). Other studies have indicated that L. longiclavatum is more frequent in insect populations occurring in the northern Canadian Rockies diminishing in southern areas of that mountain range, suggesting latitude influences the frequency of this fungal mutualists, due to its better cool temperature tolerances. Our findings suggest Colorado isolates may have a greater tolerance of warmer temperatures than those from the north. These findings also increase our knowledge about the species distribution and the in situ conditions permissive of its occurrence in areas south of the Canadian Rockies. Keywords: blue-stain, mycangia, mutualist, symbiont, phytopathogen.

Populations of mountain pine beetle (Dendroc- Fengel 1988, 1990). The relationship between MPB tonus ponderosae), hereafter MPB, have been in an and its three main fungal associates Grosmannia irruptive stage across many western North Ameri- clavigera (Rob.-Jeffr. & R.W. Davidson) Zipfel, Z.W. can pine forests since the 1990s. Since then, out- de Beer & M.J. Wingf., Leptographium longiclava- breaks have spread to unusually high latitudes in tum S.W. Lee, J.J. Kim & C. Breuil, and Ophiostoma Canada including new areas in Alberta (Culling- montium (Rumbold) Arx represents a near obligate ham et al. 2011). Populations have also reached un- mutualistic symbiosis in which these fungi obtain usually high elevations in the Rocky Mountains. In transport mainly by the MPB. Similarly, the beetle’s northern Colorado, attacks by the MPB at eleva- larvae require nutrients from the beneficial blue- tions higher than 3000 m are not common and have stain fungi to fully develop into adults (Six & Paine been discussed only in one internal report (DeLeon 1998, Myrholm & Langor 2015). 1939, unpubl.) and a single publication (Mitton & Across the USA, the occurrence of the MPB sym- Ferrenberg 2012). These expansions may be attrib- bionts G. clavigera and O. montium has been docu- uted to a warming climate. mented for several decades (Rumbold 1941, Robin- Bark beetles can carry a wide range of phoretic son-Jeffrey & Davidson 1968). However, L. longi- associates (Mercado et al. 2014, Hofstetter et al. clavatum has only recently been documented from 2015) including important mutualists that may also continental USA, first in California (Lee et al. 2005, become affected by changes in climate. Of these, the Massoumi Alamouti et al. 2011) and later from most best understood are the ophiostomatoid blue-stain of the states where the MPB occurs (Ojeda Alayon fungi. The name “blue-stain fungi” comes from the et al. 2017). The species has been frequently report- characteristic grayish blue-green hues seen on in- ed from MPB populations in Canada (Lee et al. fected wood due to refraction of incident natural 2005, Rice et al. 2008, Roe et al. 2011, among others). light by the melanin in their hyphal bodies (Zink & A caveat of previous knowledge from the USA is

Sydowia 70 (2018) 1 Mercado & Ortiz-Santana: Beetle mutualist Leptographium longiclavatum that investigations utilizing techniques capable of ing familiarized, amended media was not used for detecting this MPB fungal symbiont in the US have the rest of the study to obtain information about been used less frequently than in Canada (Ojeda other MPB fungal associates for future studies. Alayon et al. 2017). Our objective was to determine Direct cultures of dissected mycangia and ely- the main blue-stain fungal species of MPB popula- tral surfaces were grown in un-amended 2 % MEA tions affecting areas of three common host pines in petri dishes (100 mm). A direct culture method of northern Colorado, using morphological and mo- specific transporting structures of the MPB was lecular characterizations. chosen following previous studies (Whitney & Far- ris 1970, Six & Paine 1998). Within two to seven Materials and methods days after initial culture, agar plugs (2 mm) of mor- phologically different fungi were transferred to wa- Field sampling ter agar. Two to four days later, hyphal tips were To infer potential effects of temperature in blue- transferred to un-amended 2 % MEA plates to grow stain fungi diversity we instrumented trees with pure fungal strains. Colonies were grown at room temperature sensors (Onset, S-TMB-M002) in 2011 temperature (22 °C) without protection from day- and within 5 days of insect attacks to measure phlo- time fluorescent light exposure for a period of28 em temperatures during their development. These days. Replicates of pure fungal cultures were used probes were drilled into the phloem at the north for DNA extraction and to study fungal growth and south sides. In 2012 and 2013, adult flying MPB characters in wood discs. Conidia were produced were collected at different sites in the Roosevelt profusely in sterile wood discs from the MPB three National Forest in northern Colorado from a popu- pine hosts; therefore, these were used to examine lation receding from the climax of its epidemic in the type of conidia and conidiophore as well. 2011 (Figs. 1–2). Insect collections were made during mid to late July which corresponded to the bee- Morphological determinations tle’s main flight and attack period during these Observed cultural characters used to distinguish years. Bark beetles were not directly handled nor L. longiclavatum from G. clavigera were those de- did these interact with other insects as these were scribed by Lee et al. (2005). Characters used to de- induced to drop into individual sterile micro centri- termine O. montium were those defined by Rumbold fuge tubes with lid (1.5 ml, Fisher Scientific). -In (1941). Microscopic characterization using the sects were transported in coolers and refrigerated Stereomaster (Fisher Scientific) and Eclipse TS100 at the laboratory at –18 °C until processed. (Nikon) microscopes included distinguishing the asexual morphs Hyalorhinocladiella from Lep- Statistical analyses on fungal presence tographium, as well as the types of conidia pro- We compared prevalence (a proportion) which duced by the fungi. inherently accounts for differences in the number of MPBs obtained across years. We used a generalized Molecular determinations linear mixed model (GLMM) with a logit link to as- DNA sequences of the nuclear ribosomal ITS2- sess patterns in fungal presence and included MPB LSU (partial 5.8 + ITS 2 + partial 28S) and the pro- ID as a random effect to account for repeated obser- tein-coding gene β-tubulin (partial gene) were used vations (Hosmer et al. 2013). Differences between in the present study. DNA extraction and amplifica- factors (year and species) were discerned using Tuk- tion were performed at the Center for Forest Mycol- ey multiple comparisons. All statistical analyses ogy Research (CFMR) while the sequencing was per- were performed in R using the lme4 and multcomp formed at the University of Wisconsin Biotechnology packages (Hothorn et al. 2008, R Core Team 2017). Center (UWBC) in Madison, WI. DNA was extracted with a CTAB protocol following Palmer et al. (2008) Fungal culturing and Lorch et al. (2013), but with certain modifica- In 2011, the first author became familiar with tions. A small amount of mycelium from cultures the blue-stain fungi transported by the mountain was scraped using a flattened inoculation needle and pine beetle in Colorado. At first, the protocol used placed in 400 µl 2 % CTAB lysis buffer in 1.5 micro- included selective antibiotic-amended media [1 % tubes. Tubes were frozen overnight at –20 °C and Streptomycin (Sigma) and 2 % Cycloheximide then the tissue samples were ground using a plastic (Acros Organics)] that allowed the near-exclusive blue pestle. After this, the tubes were incubated in a growth of resistant blue-stain species. After becom- 65 °C water bath for two hours. The samples were

2 Sydowia 70 (2018) Mercado & Ortiz-Santana: Beetle mutualist Leptographium longiclavatum

Fig. 1. Study site (star) in the Roosevelt National Forest (gray polygon). The forest is part of the southern Rocky Mountains. – Fig. 2. Affected forest hectares in Larimer County, Colorado from 2009 to 2013 by Mountain pine beetle relates directly to the insect’ population level which was receding during the study. Most of this insect’s activity in this county was located within the Roosevelt National Forest. Data adapted from USDA/Forest Health and Technologies. then centrifuged at 13000 rcf for 6 min at room tem- other sequences available in GenBank (Benson et al. perature (RT) and 300 µl of supernatant were trans- 2013) via BLASTn search. Ten new ITS2-LSU and ferred to a new tube. Next, 350 µl of cold 2-propanol β-tubulin sequences were generated, and 27 se- was added to each tube and placed at –80 °C for quences pertaining to the genera Leptographium 15 min. Samples were centrifuged at 10000 rcf for and Grosmannia were retrieved from GenBank. The 20 min at 4 °C and then supernatant was discarded selection of sequences from GenBank was performed and 375 µl 75 % ethanol was added and tubes were considering the BLASTn search results and the spe- centrifuged at 13000 rcf for 6 min at RT. Supernatant cies grouped within the Grosmannia clade (Zipfel et was discarded and pellets were air-dried at RT for al. 2006, Roe et al. 2010) and the Grosmannia clavi- at least 5 min, then resuspended in 50 µl water. DNA gera complex (Six et al. 2011) in previous studies. was cleaned with the GeneClean III kit as indicated The information for these isolates is provided in Ta- by Palmer et al. (2008). ble 1. Newly generated sequences were edited with The ITS2-LSU region was amplified with primers Sequencher 4.8 (Gene Codes Corp., Ann Arbor, ITS3 (White et al. 1990) and LR3 (Vilgalys & Hester Michigan) and were deposited in GenBank 1990), while the β-tubulin was amplified with primers (KY940824-KY940843). DNA sequences were T10 (O’Donnell & Cigelnik 1997) and Bt2b (Glass & aligned with MAFFT 7 (Katoh & Standley 2013). The Donaldson 1995). PCR was performed using 5x Green Q-INS-I algorithm was used to align both the ITS2- GoTaq reaction buffer and polymerase (Promega, LSU and β-tubulin sequences. The alignments were Madison, WI) as indicated by Palmer et al. (2008). manually adjusted with MacClade 4.08 (Maddison Thermo-cycler conditions for the ITS2-LSU region & Maddison 2002). The final alignment was depos- were: initial denaturation at 94 °C (2 min), followed ited in TreeBASE (S20898). Phylogenetic analyses of by 30 cycles of denaturation at 94 °C (40 s), primer the combined ITS2-LSU + β-tubulin dataset were annealing at 53 °C (40 s) and elongation at 72 °C (130 performed using maximum likelihood (ML) and s); and a final extension step of 72 °C (5 min). For the Bayesian (BY) methods. Maximum likelihood analy- β-tubulin, the PCR conditions followed Gorton et al. sis was performed using RAxML black box (Stama- (2004). PCR products were sequenced in both direc- takis et al. 2008) under the GTR model with GAMMA tions with the BigDye sequencing Kit and an ABI distributed rate heterogeneity and 100 rapid boot- 3730xl capillary sequencer at UWBC. strap replicates. Bayesian analysis was performed using MrBayes 3.2.6 (Ronquist et al. 2012) on XSEDE Phylogenetic analyses through the CIPRES Science Gateway (Miller et al. DNA sequences used for molecular identification 2010) for 3000000 generations in two runs and four and phylogenetic comparison were compared with chains with trees sampled every 1000 generations.

Sydowia 70 (2018) 3 Mercado & Ortiz-Santana: Beetle mutualist Leptographium longiclavatum

The burn-in period was set to 0.25. Phylogenetic Two types of colonies were common among the trees were visualized and edited via FigTree growing cultures, those with an “entire” or a “sinu- v.1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/) ous” outer margin. Organisms with the entire outer and rooted to midpoint. margin (Fig. 6) were separated by color (i.e. sepia vs. olive-brown). Strains of the sepia colored organisms Voucher material in agar media and wood discs produced Hyalorhino- Studied specimens (Tab. 1) were isolated from D. cladiella asexual morphs (Fig. 8) and short and oval ponderosae active in the Roosevelt National Forest conidia, diagnostic characters of O. montium (Rum- in Colorado, USA. by the first author in the sum- bold 1941). The olive-brown colonies produced Lep- mers of 2012 and 2013. All isolates are maintained tographium asexual morphs (Fig. 9) in media and in the Rocky Mountain Research Station (RMRS) in wood discs. Microscopically, conidiophores pro- Fort Collins, CO and duplicates have also been de- duced long septate-clavate conidia, characters used posited at the Center for Forest Mycology Research for the diagnosis of G. clavigera (Lee et al. 2005). Culture Collection (CFMR) in Madison, WI. Ophiostomatoids with a sinuous outer margin of their colonies also differed by their dark-olive color (Fig. 7). As in G. clavigera, wood-disc subcultures of Results this third ophiostomatoid produced Leptographi- Field study um type conidiophores with long and clavate-sep- In 2012, at our mid-elevation sites and prior to tate conidia. However, this strain was distinguished MPB emergence, we registered seven periods in from G. clavigera by a greater conidial production which temperatures exceeded 30.0 °C, and theoreti- near the center of the colony, a diagnostic character cally were able to limit the growth of G. clavigera of L. longiclavatum (Lee et al. 2005). and L. longiclavatum (Fig. 3). These periods were Molecular determinations short, none exceeding a couple of hours. These temperatures were measured on the south (warmer) The combined ITS2-LSU + β-tubulin dataset side within 13 days prior our estimated MPB emer- consisted of 37 ingroup sequences with a total of gence date of July 2. 1013 characters (616 characters for ITS2-LSU and Overall, we found all three species of fungi were 397 characters for β-tubulin), including 7 species of more abundant in 2012 than in 2013 samples (Fig. 4). Leptographium and 4 of Grosmannia. The tree to- In both years, L. longiclavatum and O. montium pologies obtained from the ML and BY analyses were the most common fungi. Although their fre- were congruent therefore only the BY tree topology quency diminished nearly by half from 2012 to is presented here (Fig. 10). Three main clades 2013, there were no significant difference in their (strongly supported) were obtained in the present population prevalence in the two years (p-value = 0.71). Grosmannia clavigera was significantly scarc- er than L. longiclavatum (p-value = 0.0002) and also than O. montium (p-value = 0.0017) during both years (Fig. 5). Although 2013 was a climatically moderate year in contrast to the record warm 2012, there was no reduction on the previously considered warm-adapted O. montium (Rice et al. 2008) or an increment of the considered cold-tolerant L. longiclavatum and G. clavigera (Rice et al. 2008, Addison et al. 2013).

Morphological determinations Morphological characterization by the leading author led to the identification of three species, G. clavigera, L. longiclavatum, and O. montium, three Fig. 3. Recorded phloem temperature (°C) around the time of well known mutualistic blue-staining ophiostoma- mountain pine beetle emergence in 2012. Vertical dotted line toids of the MPB. Determinations were based on the indicates the estimated date of beetle emergence determined by examining captures from flight intercept traps in 2012 at study of both cultural and microscopic characteris- 2800m. Gray-shaded areas represent the theoretical growth tics as detailed below. advantage of O. montium over the other species.

4 Sydowia 70 (2018) Mercado & Ortiz-Santana: Beetle mutualist Leptographium longiclavatum

Tab. 1. Grosmannia and Leptographium isolates used in the study with GenBank accession numbers for sequences.

GenBank accession no. Species Isolate Location ITS-LSU β-tubulin

G. aurea CMW 438.69 Canada-BC JF798473 JF798454 UC03DL06 Canada-AB GU370267 GU370181 UC16G21 Canada-AB GU370293 GU370207 G. clavigera CO-JM-13 USA-CO KY940831 KY940841 CO-JM-17 USA-CO KY940832 KY940842 CO-JM-41 USA-CO KY940833 KY940843 SL-Kw1407 Canada-BC AY544615 AY263195 UC27G29 Canada-BC GU370261 GU370175 UC17DL22 Canada-AB GU370273 GU370187 G. huntii CBS398.77 Canada AY707208 AY707194 UAMH4997 Canada AY544617 AY349023 G. robusta CMW 668 USA-ID AY553397 AY534945 CMW 2805 USA-ID AY553396 AY534944 L. koreanum DUCC 3020 Korea KR012411 KR012443 MUCL 46362 China-Shanxi EU502799 EU502811 L. longiclavatum CO-JM-8 USA-CO KY940824 KY940834 CO-JM-37 USA-CO KY940825 KY940835 CO-JM-42 USA-CO KY940826 KY940836 CO-JM-43 USA-CO KY940827 KY940837 CO-JM-44 USA-CO KY940828 KY940838 CO-JM-45 USA-CO KY940829 KY940839 CO-JM-46 USA-CO KY940830 KY940840 SL-Kp11 Canada-BC AY816687 AY816712 SL-Kw1436 Canada-BC AY816686 AY288934 SL-Pw5 Canada-BC AY816689 AY288935 UL02G20 Canada-BC GU370299 GU370213 UL13G22 Canada-BC GU370300 GU370214 UL02G23 Canada-AB GU370262 GU370176 L. lundbergii CMW 217 Sweden DQ062065 AY707185 UAMH 9584 Sweden AY544603 AY263184 L. procerum MUCL 46323 China-Shanxi EU296775 EU296781 MUCL 47244 China-Shanxi EU502796 EU502807 L. terebrantis ATCC 58098 USA-MN JF798476 JF798460 CBS337.70 USA-LA JF798477 JF798459 L. wingfieldii CMW 2095 France AY553400 AY534948 CMW 2096 France AY553398 AY534946 L. yunnanense CMW 5152 China-Vimen AY707207 AY707193 study. The first main clade includes three subclades, and consisting of isolates of G. aurea (Rob.-Jeffr. & the L. longiclavatum clade (strongly supported and R.W. Davidson) Zipfel, Z.W. de Beer & M.J. Wingf. consisting of L. longiclavatum isolates from Cana- and L. terebrantis S.J. Barras & T.J. Perry]. The sec- da and Colorado, USA), the G. clavigera clade [not ond main clade includes two subclades, the L. kore- supported and consisting of isolates of G. clavigera, anum clade (supported in the BY analysis, consist- G. robusta (Rob.-Jeffr. & R.W. Davidson) Zipfel, ing of isolates of L. koreanum J.J. Kim & G.H. Kim, Z.W. de Beer & M.J. Wingf., and L. wingfieldii M. L. yunnanense X.D. Zhou, K. Jacobs, M.J. Wingf. & Morelet], and the G. aurea clade [strongly supported M. Morelet and L. lundbergii Lagerb. & Melin and

Sydowia 70 (2018) 5 Mercado & Ortiz-Santana: Beetle mutualist Leptographium longiclavatum

Fig. 4. Predicted probability of species prevalence between the two years. – Fig. 5. Results of linear multiple comparisons on logit scale with corresponding 95 % confidence intervals; confidence intervals which overlap zero (horizontal dashed line) indi- cate no significant difference. Abbreviations are “L.lon” for L. longiclavatum, “G.cla” for G. clavigera, and “O.mon” for O. montium.

the G. huntii clade [consisting of G. huntii (Rob.- clade in previous studies (Zipfel et al. 2006, Roe et Jeffr.) Zipfel, Z.W. de Beer & M.J. Wingf. isolates]. al. 2010, Six et al. 2011, De Beer & Wingfield 2013). The third main clade includes isolates of L. procer- Unexpectedly the usually considered “cold- um (W.B. Kendr.) M.J. Wingf. and appears not close- adapted” L. longiclavatum was as common in MPB ly related to the other two. mycangia as the “warm-adapted” O. montium during 2012. This year was the warmest recorded year Discussion in Colorado reaching 3.6 °C above average (NOAA 2012) in late June, just before the MPB peak emer- Using morphological and molecular characteri- gence in that year. At the moderate elevation site zations we identified three species of blue-stain (2800 m), the warmest phloem temperature was re- fungi from MPB collections in Colorado. These spe- corded on June 25 (30.3 °C). Despite being close to cies represent Grosmannia clavigera, Leptographi- Canadian isolates of G. clavigera and L. longiclava- um longiclavatum, and Ophiostoma montium. Iso- tum which growth is stunned or stops (Solheim & lates of O. montium were not included in the phylo- Krokene 1998, Rice et al. 2008) near this tempera- genetic analyses in the present study, but ITS and ture, USA isolates of G. clavigera have been found ITS-LSU BLASTn queries indicated that the near- to sporulate abundantly at 30 °C (Moore & Six est matches represented strains of O. montium (ex- 2015). Although the same has not been tested for ceeding 97 % similarity with isolates CMW 13220, isolates of L. longiclavatum, in Canada its isolates CMW 1321 and WIN 1635). Isolates of G. clavigera sporulate at higher temperatures than those of G. and L. longiclavatum from this study grouped with clavigera (Rice et al. 2008). Therefore, is likely that those from Canada (Fig. 6), confirming the identity L. longiclavatum isolates from Colorado and other of these species and the presence of L. longiclava- southern latitudes are not only adapted to warmer tum in Colorado. The phylogenetic analyses also temperatures but also may be able to sporulate at suggest the differentiation between Grosmannia similar temperatures than sympatric G. clavigera and Longiclavatum species, grouped within the phenotypes, suggesting a life history highly syn- Grosmannia clavigera complex or Grosmannia chronized to MPB in these latitudes.

6 Sydowia 70 (2018) Mercado & Ortiz-Santana: Beetle mutualist Leptographium longiclavatum

Figs. 6–7. The “entire” margin Ophiostoma montium (6) colonies contrasting with the “sinuate” (branching) margin of (7) Lep- tographium longiclavatum cultures. Photographs J. E. Mercado.

Moreover, recent studies that independently ex- two blue-stain mutualists occurring with this in- amined fungi at one of our study sites determined sect. Competitive interactions between L. longi- that some L. longiclavatum isolates achieved nor- clavatum and G. clavigera result in a higher fre- mal growth rate at 30 °C in Colorado (Ojeda Alayon quency of L. longiclavatum in the Canadian Rock- et al. 2017). These temperatures also allow the nor- ies due to its cooler temperature tolerance (Rice et mal growth of O. montium (Solheim & Krokene al. 2008, Ojeda Alayon et al. 2017), or due to latitu- 1998, Rice et al. 2008). In 2012, we found no thermal dinal factors (Roe et al. 2011). Nevertheless, our re- advantage favoring the growth of the warm-adapt- cord of phloem temperatures indicates that condi- ed O. montium within the month before emergence tions fostering the growth of the three blue-stain were temperatures exceeded 30 °C occasionally. The mutualists at moderate and high elevation pine for- high frequency of L. longiclavatum in Colorado and ests in the southern Rockies of Colorado exist even its presence in other areas south of the Canadian during extremely warm years and that their preva- Rockies suggests its association with the MPB may lence may be driven by other factors. At this lati- be as important and prevalent as that of the other tude (43 N), temperatures uniquely permissive for

Figs. 8–9. The Hyalorhinocladiella asexual morph (8) of O. montium is easy to distinguish from the Leptographium asexual morphs (9) of both L. longiclavatum and G. clavigera. The clavate-septate conidia of the latter are unique of the American biome in the Order Ophiostomatales. Photographs J. E. Mercado.

Sydowia 70 (2018) 7 Mercado & Ortiz-Santana: Beetle mutualist Leptographium longiclavatum

Fig. 10. Bayesian consensus tree based on combined ITS2-LSU+β-tubulin sequences. Support values along branches are from ML bootstrap (>65 %) and BY analysis (PP >0.95). the growth of O. montium (i.e. >30) may be uncom- with equal repercussions for its ecologically and mon. economically important insect carrier. It also, rep- The matching frequencies of L. longiclavatum resents one of the firstin situ tests of previous theo- and warm temperature adapted O. montium during retical predictions complementing and supporting the warmer year on record suggests that currently findings from in vitro experiments about the devel- there is no temperature barrier for the prevalence of opment of MPB fungal mutualists in this symbiosis. L. longiclavatum in moderate to high elevation pine forests of Colorado. Furthermore, reports of the species from AZ (Ojeda Alayon et al. 2017) suggest Acknowledgements L. longiclavatum could potentially tolerate even warmer temperature in western USA. Further re- The authors thank Dr. Daniel L. Lindner (NRS) search should explore variations across larger geo- for his critical review of the manuscript and Shan- graphical scales to account for geographical pheno- non Kay (RMRS) for statistical support. This work typic variability of the fungal associates of this im- was funded by the Rocky Mountain Research Sta- portant insect. Our findings greatly improve the tion and Northern Research Station of the USDA/ knowledge of the adaptive capacity of this species, Forest Service.

8 Sydowia 70 (2018) Mercado & Ortiz-Santana: Beetle mutualist Leptographium longiclavatum

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